The invention relates generally to the field of signal transduction and, more specifically, to compositions and methods for indicating properties of membrane molecules using fluorescence resonance energy transfer (FRET).
The transduction of signals from the outside to the inside of a cell underlies most cellular processes, including proliferation, differentiation, apoptosis, motility and invasion. Therefore, there is considerable interest in developing improved methods of monitoring signal transduction in response to normal and abnormal stimuli. Methods of monitoring signal transduction have numerous applications, such as in identifying or improving modulators of signal transduction pathways, which are candidate therapeutic drugs or therapeutic targets, and in detecting pathological alterations in cells.
Some of the earliest and most sensitive signals transduced in response to stimuli involve changes in properties of membrane molecules, including membrane lipids and polypeptides, such as changes in location, abundance, conformation or post-translational modification state. Accordingly, there exists a need to develop compositions and methods suitable for indicating changes in properties of membrane molecule.
An early response to agonist stimulation of many tyrosine kinase and G-protein coupled receptors is the activation of the enzyme phospholipase C, which cleaves the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate second messengers that increase cytosolic free Ca2+ concentration. Although Ca2+ indicators and methods have been described that allow monitoring of Ca2+ concentration in single living cells with high spatial and temporal resolution, Ca2+ fluxes, being more distal to receptor activation, may not as faithfully report receptor activation levels as changes in PIP2 levels.
In a recently developed method for detecting PIP2 dynamics in living cells, a pleckstrin homology (PH) domain tagged with a green fluorescent protein (GFP) has been used. Detection of PIP2 hydrolysis was by in vivo visualization, such as by confocal imaging and post acquisition image analysis, of translocation of the fluorescence from the membrane to the cytosol. However, this method suffers from several disadvantages. First, it is hard to obtain quantitative data using confocal microscopy, since even minor focal drift and changes in cell morphology that often occur after stimulation render quantitative measurements unreliable. Second, it is difficult to visualize translocation in very flat cells or in cellular subregions. Third, at fast imaging rates, confocal imaging requires high excitation intensities that can cause severe cell damage in minutes. Fourth, the imaging approach is not easily extended to cell populations. Therefore, there exists a need to develop improved methods for detecting PIP2 dynamics in cells, and particularly methods amenable to high-throughput screening.
The present invention satisfies these needs and provides related advantages as well.
The invention provides a phosphatidylinositol 4,5-bisphosphate (PIP2) indicator. The indicator contains:
(a) a first polypeptide having:
(i) a pleckstrin homology (PH) domain; and
(ii) a donor fluorescent domain
(b) a second polypeptide having:
(i) a pleckstrin homology (PH) domain; and
(ii) an acceptor fluorescent domain;
wherein fluorescence resonance energy transfer (FRET) between the donor domain and the acceptor domain indicates PIP2 levels.
Also provided is a nucleic acid kit, the nucleic acid molecule components of which encode a PIP2 indicator, the indicator containing:
(a) a first polypeptide having:
(i) a PH domain; and
(ii) a donor fluorescent domain
(b) a second polypeptide having:
(i) a PH domain; and
(ii) an acceptor fluorescent domain;
wherein fluorescence resonance energy transfer (FRET) between the donor domain and the acceptor domain indicates PIP2 levels.
Further provided is a method of indicating PIP2 levels in a cell. The method includes the steps of:
(a) providing a cell containing a PIP2 indicator; and
(b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain, wherein FRET between the donor domain and the acceptor domain indicates PIP2 levels in the cell.
The invention also provides a method of identifying a compound that modulates PIP2 levels in a cell. The method includes the steps of:
(a) contacting a cell containing a PIP2 indicator with one or more test compounds; and
(b) determining FRET between the donor fluorescent domain and the acceptor fluorescent domain following the contacting,
wherein increased or decreased FRET following contacting indicates that the test compound is a compound that modulates PIP2 levels in the cell.